Postsynaptic-gabaergic inhibition of non-pyramidal neurons in the guinea-pig hippocampus

POSTSYNAPTIC-GABAERG~C INHIBITION OF NON-PYRAMIDAL NEURONS IN THE GUINEA-PIG HIPPOCAMPUS U. MISGELD* and M. FROTSCHER~ *Department of ~e~rophysi~i~~y, Max-Planck-lnstitut fiir Psychiatric, Am Kfopferspit7 IXA. I)-Xti3.I Planegg-Martinsried and tInstitute of Anatomy, Johann Wolfgang ~nethe-~niv~rslt~it. Il.hO(tti Frankfurt/Main, F.R.G. Abstract-Intracellular recording and staining was applied to study non-pyramidal neurons II: illi. guinea-pig hippocampus. To avoid accidental impalement of pyramidal or granule cells. two h~ppocamp:~l regions known to be devoid of pyramidal or granule cells were chosen. In transverse and longitudinal slices. neurons of the deep hilar region (zone 4 of Amaral’), and in transverse siices, neurons of the stratum l~lcunosum-mole~ulare (CA3) were impaled. The intra~l~ular staining with Lucifer Yellow rcvcaled that of 20 neurons stained in these zones all were non-pyramidal neurons. Mar neurons, situated just below the granular layer, differed from granule ceffs and CA3 neurons with respect to their action potential waveform and their current/voltage relationship. In contrast to granule cells, hitar neurons exhibited spontaneous bursts in the presence of bicuculline (25 IIM)_ In ali neuronr impaled in the hilar region and the stratum lacunosum-moleculare (n =42), inhibitory postsynaptIc potentials could be elicited. These inhibitory postsynaptic potentials were blocked by hicuculltne. 1~

transverse slices, perforant path stimulation elicited inhibition preceding excitation in hilar ncur
Gamma-aminobutyric

acid (GABA)

is considered

the

cells electrophysiologically

have

focused

on

jllxt~l-

pyramidal zones.~~22~36~” However. the yield has been main inhibitory transmitter in the mammalian hipporather low2’ and the stability of recording limited. campal formation.” Candidates for GABAergic neuA better yield has been obtained by usmg lortpirons :irc non-pyramidal cells, since pyramidal cells and dentate granule cells have been found to be tudinal rather than transverse hippocampal shccs.” In our hands, the yield was, reduced by the fact that excitatory.” Accordingly, only non-pyramidal neulight microscopic examination after intracellular rons have been immunostained by antibodies against staining revealed pyramidal neurons within the GABA”,4’ or its synthetizing enzyme glutamate decarboxylase’h.“.3?.39.40 (GAD). Although being a pyramidal layer even if the recording electrode had been clearly located outside the pyramidai layer This minority compared to pyramidat cells in the hipcan be explained by staining via thick pr<,ximal pocampus proper (approximately 12%; Dietz and dendrites of pyramidal cells in stratum orlcns or Frotscher, unpublished observations), non-pyramidal stratum radiatum. Another pitfall was that pyra~nldal neurons seem to play an important role in informaneurons were found in atypical positions cmtaide the tion processing. since they have been implicated in recurrent inhibition,~,‘,~’ feed-forward inhibitionJ.4,9,13 zone visually identified as the pyramidal layer in the slice. Despite these problems, it is of course, an and disinhibition.” advantage of the slice preparation that electrodes can In immunocytochemical studies,‘6.3J,32.39.40 GADbe positioned under visual guidance. Thus it is possiimmunoreactive neurons were found in all hippoble to select zones for recording where an impalement campal layers with neurons just above and below the pyramidal and granular layers predominating. AC- of a pyramidal cell or a granule cell is at least very unlikely. cordingly, previous attempts to characterize these For this purpose we chose the stratum lacunosum-moleculare (CA3) and the hilar region Abbreriations: EPSP, excitatory postsynaptic potential; (zone 4 of Amara13). Of the pyramidal cells, the GABA, y-aminobutyric acid; GAD, glutamate decarstratum lacunosum-moleculare contains only thin boxylase; IPSP, inhibitory postsynaptic potential; PAP, dendritic branches unlikely to be impaled (cf. Fig. I ). peroxidase-antiperoxidase; LY, Lucifer Yellow.

193

U. MISGELDand M. FROTSCHER

t94

Amaral’s’ zone 4 was chosen because this area contains many GAD-positive cell~.~‘~~*In addition, the

adjacent granule cells lack basal dendrites extending into the hilus. Naving established by intracellular staining that non-py~midal neurons and no pyramidal or granule cells were impaled, we detailed electrophysiological properties of cells in these zones. Special attention was directed towards their postsynaptic potentials to verify the GABAergic innervation by inhibitory synapses on these presumably inhibitor neurons. Further, the possible GABAergic innervation of GABAergic neurons in these zones was studied by immunocytochemistry. EXPERIMENTAL

PROCEDURES

Siiee preparation

Guinea-pig hippocampal slices were prepared by handcutting as described previously.” For the present study the

hippocampus was not only sliced in transverse, but also in lontritudinal direction (Fig. 1). This change was made be&use Knowles and &hwa&kroit?* encountered nonpyramidal neurons more frequently in longitudinal slices than in transverse slices. The reason for this may be a better preservation of inhibitor neurons which exhibit a longitudinal, interlamellar orientation.6 After dissection of the hippocampus, both ends were cut away and the remaining middle part was cut into 3-4 blocks. Of these blocks, the region inferior was trimmed away and, subsequently. 200-300 pm thick slices were cm from the trimmed surface. Only those slices were chosen for recording in which CA1 pyramidal cells and the superior (lateral) and inferior (medial) blades of the fascia dentata were clearly visible (Fig. IB). Care was taken that they did not show any hint of the presence of the terminal extention of the pyramidal layer in regio inferior (CA4). Moreover, recordings were restricted to a zone directly beneath the granular layer (polymorphic layer) where no pyramidal cells or modified pyramidal cells occur? During preincubation and recording the slices were kept in an oxygenated (95% 4, 5% CO,} buffer which was composed of (in mM): NaCi, 124; KCI,

or PYr

rad

lac

POJ; rad

R 9 h

P Fig. 1. Recording area. Schematic drawing demonstrating the methodical approach. (A) Non-pyramidal neurons in the hippocampus proper were impaled in stratum lacunoaum-molecuhrre (CA3) of transveme slices. This zone is far away from the per&arya and thick pmxixnd dcndritosof pyramidal neurons. (3) Non-pa neurons in the h&u region were impaled just t&emeath the gramrle ceil layer. This was done in transverse (left) and Iongitudinal (right) slices of the h@ocampus. Asterisks indicate typical position of the mcording &ctro&. Longitudinal alices were obtained after cutting away the fimbria and regio inferior as shown in the left drawing. Lon&dinalshces prepared this way (right) acoo&& exhibit the CA1 pyramidal layer and the supmpymmW and infbapymmW blades of the fascia dentata but no pyramidal neurons of regio inferior. fh, hippocampal fissure; g, gmnule cell layer; h, hilus; lac, stratum lacunosum-mole&are; m, dentate molecular layer; or, stratum oriens; pyr, stratum pyramidale; rad, stratum radiatum.

Fig. 2. lntracellularly stained hippocampal neurons. (a) Pyramidal neuron in CA3. (b) Granule cell in the suprapyramidal blade of the fascia dentata. (c) Non-pyramidal neuron in stratum lacunosum-moleculare of CA3. Pyramidal cell layer at the bottom of the figure. (d) Non-pyramidal neuron in the hilar region underneath the granule cell layer. An arrow points to the axon which can be traced towards its cut end. (ax) Transverse slices of the hippocampus. (d) Longitudinal slice. x 140.

19.5

Fig. 3. Intracellularly stained non-pyramidal neurons. (a) Multipolar non-pyramidal neuron in the stratum lacunosum-moleculare (transverse slice). Pyramidal ceil layer at the bottom of the figure. An arrow points to the axon which bifurcates and crosses the outer segments of apical pyramidal cell dendrites passing throttgh that zone. x 140. (b) Non-pyramidal neuron with predominantly horizontal dendrites in the area dentata directly beneath the granule cell layer. This cell was impaled in a longitudinal slice of the hippocampus (see c for comparison). x 140. (c) Large horizontal non-pyramidal neuron located just below the granule cell layer. Area dentata in a transverse slice. x 140. (d) Varicose dendrite of the neuron shown in (a). x 830. (e) Spiny dendrite of the neuron shown in (c). x 830. 196

197

Fig. 9. Light (a) and electron micrograph (b) of GAD-immunoreactive neurons and terminals in the area dentata. (a) Three immunoreactive neurons are shown which are located just underneath the layer of granule cells (g). Note immunoreactive puncta surrounding the non-immunoreactive cell bodies of granule cells, It appears that the immunoreactive terminals also contact the GAD-positive perikarya (arrowheads). x 1200. (b) GAD-positive horizontal non-pyramidal neuron located just below the granule cell layer. A GAD-immunoreactive terminal forms a symmatric synaptic contact (arrow). x 36,000.

Fig. 8. Epileptiform activity of a hilar non-pyramidal neuron in a transverse slice during staining in the presence of bicuculline (25 pm). (a) The fluorescence micrograph of this neuron which was found to be situated underneath the granule cell layer ( x 200, neuron displayed also in Fig. 3c in a different focus and at different magnification). (b) Synaptic response of this neuron to perforant path stimulation. The cell had been impaled in the same slice and in the immediate neighborhood of the cell shown in Fig. 7A. The cell was impaled after the slice had been bathed in bicuculline for 1 h. The response was taken during the injection of hyperpolarizing current (0.5 nA) for dye injection. (c. d) Spontaneous burst. Despite current injection for staining, the neuron burst spontaneously, however, at a rather low frequency. In (c) the spikes are truncated. In (d) the scope was triggered late on the rising phase of the depolarizing envelope to show the full sized spikes. There was only little decrement of spike amplitude during the burst. Voltage calibration applies to all traces.

198

Non-pyramidal

neurons

5.1: MgSO,, 1.3; KH,PO,. 1.22; NaHCO,, 25.5; CaCI,, 2.5: glucose. 10. The pH was 7.4 and the temperature was 36 C. During some experiments C+ )-bicuculline (25 PM, Sigma) was added to the standard buffer.

For electrical stimulation (0.5 Hz, 5- 30 V, 0. I a.2 ms) bipolar electrodes were positioned onto the surface of the slice to activate m transverse sections (i) perforant path tibers in the dentate molecular layer and (ii) local afferents to stratum lacunosum-moleculare neurons. In longitudinal section\ hilar neurons subjacent to the upper blade of the granule cell layer (Fig. I B, asterisk) were activated by local stimulation underneath the granule cell layer. Intracellular potentials were monitored with 0.6 M K,SO, containing electrodes (5G-80 MR). Intracellular potentials were fed into a neurodata (IR-283) preamplifier with an active bridge circuit, displayed on an oscilloscope and stored on magnetic tape. Data were further analyzed on a digital storage oscilloscope (Nicolet Explorer III) and printed for photography. For intracellular staining glass pipettes were filled with 8”/0 Lucifer Yellow-CH (LY, Sigma) in I M LiCl or Li,SO, (SO-100 MR). The dye was injected by negative current (0.5~ I nA) for 5 IOmin.

Following intracellular staining the slices were left in the recording chamber for at least IO min. Thereafter, they were fixed in 4% formalin for 5 min. The slices were then briefly rinsed in distilled water, dehydrated in an ascending series of ethanol, cleared in xylene. and mounted in DPX. The intracellularly stained neurons were studied under a Zeiss fluorescence microscope equipped with appropriate filter combinations.2h

Immunocytochemiccri method.? TWO guinea-pigs were perfused

transcardially under ether anesthesia with 70 ml of saline followed by a fixative contaimng 4% paraformaldehyde. 0.08% glutaraldehyde and 15”/0 saturated picric acid in 0.1 M phosphate buffer4’ (pH 7.3). The brains were removed from the skull and the hippocampi were dissected out. Tissue blocks about 3 mm thick were cut perpendicular to the longitudinal axis of the hippocampus and were stored in glutaraldehyde-free fixative overnight. Next, Vibratome sections (40pm) were prepared and washed for 24 h in several changes of phosphate buffer. Prior to immunostaining, the vials con; taining the sections in phosphate buffer were briefly frozen in liquid nitrogen, thawed to room temperature, and again washed in phosphate buffer. Following preincubation for 30 mm in 10% normal rabbit serum the sections were incubated for 48 h at 4’C in sheep antiserum S3 against

GAI>‘9 (I :2000). Immunostaining was performed with the peroxidase-antiperoxidase (PAP) technique43 which included incubation in rabbit antirat immunogammaglobulin (I :40 for I.5 h) and in sheep PAP complex (I :40, 2 h). Between each incubation step the sections were washed in several changes of phosphate buffer. The tissue bound peroxidase was visualized by incubating the sections with 3.3’ diaminobenzidine (0.07%) and H,O, in Tris buffer (0.1 M. pH 7.6) for 5-10min. In control experiments the sections were treated in the same way except that the primary antiserum was omitted. No immunostaining was observed in these cases. Following postfixation in osmium tetroxidc (1% in 0.1 M phosphate buffer for 30 min) the sections were dehydrated (block-stained with uranyl acetate in 709/o ethanol) and flat-embedded in Araldite. GADimmunoreactive cells in the hilar region and stratum lacunosum-moleculare were selected, photographed (Fig. 9a) and drawn, and re-embedded for ultrathin sectioning. The thin sections were mounted on slot grids coated with Formvar film. and studied in a Siemens 101 Elmiskop.

in the hippocampus

IY9 RESULTS

Morphological neurons

nhscrl~ations on it~traceilularl~’ stcrined

Intending to impale non-pyramidal neurons exclusively, we have chosen the stratum lacunosummoleculare of CA3 and the hilar region for recording (Fig. I). This approach seemed to be appropriate because. of 20 neurons intraccllularly stained with Lucifer Yellow (LY) in these two zones. all were found to be non-pyramidal cells. Examples are presented in Figs 2c.d. 3 and 8a. I-‘or comparison. two principal cells of the hippocampal formation. a pyramidal neuron (Fig. 2a) and a dentate granule cell (Fig. 2b) are shown. Criteria for the identification of non-pyramidal neurons were (i) the location of the cell body outside the pyramidai and granular layers, which was confirmed by fluorescence microscopy, (ii) the shape of the cell body. which was ovoid or multipolar (Figs 2c,d and 3a-c). tn contrast to the characteristic triangular, elongated pyramidal cell perikaryon (Fig. 2a), and (iii) the characteristic arrangement of the dendritic tree. In contrast to pyramidal cells, the dendrites of non-pyramidal neurons were found to arise from all parts of the cell body (Figs 2c.d and 3a-c). Differentiation of hilar nonpyramidal cells from granule cells was facilitated by the regular. unipolar and cone-shaped arrangement of the dendritic tree of granule cells (Fig. 2b). Stained hilar non-pyramidal neurons had dendrites preferentially oriented parallel to the granule cell layer (Fig. 3b,c). Axon collaterals of two hilar neurons were observed to enter the granular layer. In the stratum Iacunosum-moleculare (CA3). both horizontally (Fig. 2c) and vertically (Fig. 3a) oriented cells were stained. Their dendrites exhibited varicose swellings (Fig. 3d) and, occasionally, spine-like protrusions. In some instances it was possible to correlate the morphology of stained neurons to specific cell types as they were described in Golgi studies.’ ’ “x’ The cell shown in Fig. 3c, which was impaled in a transverse slice and was located directly beneath the granular layer, resembled the fusiform cell described by Amaral.’ It had long spine-bearing dendrites (Fig. 3e) that paralleled the fascia dentata hilus border. In the stratum lacunosum-moleculare (Figs 2c and 3a). intracellularly stained neurons were similar to horizontal and vertical non-pyramidal neurons described by Cajal”” and Lorente de No’@” (cf. cells G and H in Cajal’s Fig. 473). The neuron shown m Ftg. 3a corresponds to the non-pyramidal cells with ascending axon.“” I*’

Membrane

properties

of non-pyramidal

neurons

Having established that all cells stained in the two regions were non-pyramidal, we characterized neurons in these regions electrophysiologically using K,SO, electrodes. This approach was chosen, because LY as well as Li ’ could affect intracellular responses and since. on the other hand. extensive electro-

3

Fig. 4. Current-voltage ~~ti~~~ of a ~~~~~~ neuron (A] and a granule ceh (3). (A) H&r aon-pyramidai ceil in a ~ongitndinaI s&z. The cnrrent-v&age relationship (If was tested shortly after i~~~e~t at the initial resting abide potential of -60 mV. ~nt-vo~~~ relation was retested tater (2) when t&eresting tnemhrane potenti& had increased ~n~~~y to -7onlv, The ir&cticn of ~e~~a~z~ng current pubes of increasing ~~~ failed to dep&&e the membrane sppreciabiy (I). This couid have been due to the spike aftcrhyperp&rixations, It was, however, due to outward rectification as it became evident at the higher resting membrane potentiai (2). Sun&m@ responses (3) to the injection of constant current pulses of identical amplitude reveakd outward and inward rectification. In this test, inward and outward current pubes, 70 ms in duration, were injected at the resting membrane potenti& and &ring ~~~a~~~n and Harmon of the c&lby the iejection of fang eutrentpulses~~tfre~e~~tof~tasinfcctedfortheshort~.o~~txllina transverse slice showing inward meiifhtion w&ii&is FyEdcafor CA3 neurons and granuk ah& In this f&ure, upper traces are the vohage recordings and hnver traces the cnrrent monitor.

physjolc~~~ test@ impairsstaining.A de&&d fiterature is available on membrane properties of hi~~~~~ Pravda ceU~‘~~‘*~*“~~and dentate granuie cefh~‘~“~**’Therefore, our primary interest was to search for characteristics different from those of pyramidal end granule cells which wouid evmttmily allow us to recognize these neurons by their efectricai and synaptic responses. Stab& conings with resting membrane ~ten~~~s of at least - 55 mV and spike amplitudes of at least 7OmV (mean vague for rest&~ membrane potentj# -62mV, SD f 5.3, for action poregtiaf ruaplEhrdt 80 mV & 9.9, n = 12) could k obtainad from h&n neurons. No ckarcut d%kence in tk qnality of

reeordiggs was observed between ~~~~tu~~~ arid transverse sections. Also, the rn~b~ne properties of Mhtr non-madam neurons were not different in longitudinal and transverse &es. Therefom these cells were pooled for the description of elec&icaf membrane properties, whereas they had to be deah with separately for the description of postsynaptic potentials (see below]. The input resistar~~ of hi& neurons was measured from the steady state response to 0.1 nA byperpohuizing current pulses. The average iaput~~Obtaiaed(4Z~SQf~*~roP:?;i is in the! r6Wge of input resistances described for pyramidal and granule cetfs.‘~ Pyi&nid@ a& granuk? (cf. Fig. 4B) cells exhibit non-ohmic beha+

Non-pyramidal

neurons in the hippocampus

ior at potentials positive with reference to the resting potential. This was also true for non-pyramidal neurons, however, in contrast to pyramidal and granule cells, the current-voltage relation of hilar neurons showed a decrease of the input resistance with depolarization indicating outward, rather than inward rectification (Fig. 4). In some hilar neurons we noted a time-dependent sag of the voltage response to hyperpolarizing current steps, as has been described for hippocampal neurons. 3oTherefore it is probable that non-pyramidal neurons in the hilus possess a form of additional inward rectification (Fig. 4A 2, 3). Action potential waveform was very similar to that found for non-pyramidal neurons in juxta-pyramidal zones of CA1.37 Thus, the spike was short, i.e. below 1 ms, and displayed a prominent afterhyperpolarization (Figs 4A, 5A,B) which lasted SO-100 ms. In corresponding potential ranges, CA3 neuron and granule cell action potentials are followed by afterdepolarizations”.” (cf. Fig. 5C,D). In many neurons, small depolarizing currents of about 0.1 nA

201

were sufficient to elicit action potentials (Fig. 5A). However, this low value as well as the presence of spontaneous discharges was due to rather low resting membrane potentials (cf. Fig. 4A I, 2). The cells could be driven by current injection to fire repetitively with little decrement. We did not succeed in obtaining stable (more than 5 min) recordings from neurons in the stratum lacunosum-moleculare. The cells were always depolarized when impaled, and their spike amplitudes were low because of injury during impalement. Most neurons in this region discharged spontaneously. The quality of our recordings in this region was not sufficient to allow reliable testing of their cell properties. Postsynaptic

inhibition

In various reports (for a review see Refs 4, 6 and 13) it was noted that presumed “interneurons” rcspond to afferent stimulation with repetitive and high-frequency firing. This could result from a

2

25 niV /Pi

I

Fig. 5. Spike afterpotentials of hippocampal neurons. (A) Afterhyperpolarization in a hilar non-pyramtdal neuron beneath the granule cell layer (longitudinal slice). Resting membrane potential of the cell was - 65 mV. (1) Injection of + 0.05 nA constant current pulses 1SOms in duration. The depolarizing pulse triggers an action potential followed by a long-lasting afterhyperpolarization. (2) Short (5 ms) current pulses reaching just threshold were injected. Two responses are superimposed; in only one of them a spike was triggered. Arrow points to the afterhyperpolarization. The upper trace just shows the passive decay of the voltage gradient following the charging of the membrane. (B) Afterhyperpolarization in a hilar non-pyramidal neuron beneath the granule cell layer (transverse slice). Same approach as in (A2). Duration of the current pulse was 2 ms. The spike was triggered at the resting membrane potential (I) and during the hyperpolarization of the cell to -70 mV by the injection of constant direct current (2). The latter response can be compared directly to the responses of the cells shown in (C) and (D). (C) Afterdepolarization in a CA3 neuron and (D) in a granule cell at their respective resting membrane potentials (CA3: - 72 mV, granule cell: - 75 mV). Same test as in (A2). Voltage cahbratrons are the same in (BI) and (2) as well as in (C) and (D). Spikes in (A2), (B2). and (D) truncated.

U. MISGELDand M. FROTSCHER

202

specific membrane property and/or strong excitation or weak inhibition. Indeed, in previous intracellular studies,9.37 only small inhibitory postsynaptic potentials (IPSPs) were observed to follow the initial strong excitation. We, therefore, focused on the properties of IPSPs in non-pyramidal neurons. Local stimulation elicited in all stratum lacunosum-moleculare cells (n = 11) a prominent hyperpolarizing IPSP which was, of course, partly due to the low membrane potential of these cells. However, well polarized hilar neurons also exhibited hyperpolarizing IPSPs. In longitudinal sections, hilar neurons could be best activated by local stimulation underneath the granule cells about 0.5 mm away from the recording site. Stimulation at low intensities elicited in all cells (n = 15) pure IPSPs (Fig. 6a) which were blocked by bicuculline (25 PM, Fig. 6~). With higher stimulus intensities, the large IPSP was preceded by an excitatory postsynaptic potential (EPSP) (Fig. 6b). In transverse slices, non-pyramidal neurons in the hilus could be activated by perforant path stimulation. Low intensity stimulation of the perforant path elicited IPSPs in hilar neurons (n = 10) which, however,

Con

Bit

contained an EPSP component. This EPSP increased in amplitude and its latency shortened when the stimulus intensity was increased. Also at high stimulus intensities the IPSP preceded the EPSP; it could, however, be masked at the resting membrane potential and only become evident when the cell was depolarized by current injection (Fig. 7A). The IPSP/EPSP sequence was very characteristic for the response of non-pyramidal neurons to perforant path stimulation. In contrast, such a sequence was never seen in the responses of granule cells to perforant path stimulation (Fig. 7B). Instead, they responded with an EPSP/IPSP sequence, the latency of the initial EPSP corresponding to the latency of the initial IPSP of the hilar neurons. Bicuculline (25 PM) blocked the IPSP, and a strong excitation remained (Fig. 7A). In the presence of bicuculline, hilar neurons regularly exhibited a property which separated them from the adjacent granule cells. Stimulus-evoked epileptiform events could be observed in both transverse (Fig. 7A 5, 8) and longitudinal sections. In both, transverse and longitudinal sections, non-pyramidal

.60ms

spontaneous

Fig. 6. Synaptic activation of a hilar non-pyramidal neuron by local stimulation in a longitudinal slice. In control (Con) low intensity stimulation (a) evoked a pure IPSP and high intensity stimulation (b) an EPSP/IPSP sequence. The stimulation was applied during the injection of f0.1 nA constant current pulses. Stimulation can be recognized from the vertical artifact during the first part of the voltage transients. The stimulation artifact (in (a) preceded by a directly triggered spike) is followed by the syneptic response which is intermingled with the voltage response to current injection. In the presence of bicuculline (25 #M) the inhibitory component of the synaptic response was completely blocked (c,d). In(d) the current injected was increased to 1 nA to show that, indeed, no IPSP was left. For comparison, in (e) the response to the current injection alone is shown. In (f) a spontaneously occurring burst is seen. This burst did not occur until the neuron had been exposed to bicuculline for 45 min. whereas the IPSP had been already blocked 15 min after commencmg bicuoullme perfusion. Time calibration in (c) applies to all traces except (f), voltage calibration in (b) for (a,b,c) and (f) and in (e) for (d) and (e).

Non-pyramidal

neurons in the ~~ppo~arn~us

neurons exhibited spantaneous bursts triggered from depolarizing potentials when exposed to bicuculline for prolonged time periods (Fig. 6f, 8). This behavior, while also seen in CA3 neurons is never observed in granule

~e‘tls.~~~~~

~~~~~~~~~~~~~~r~~.~.~~~e j~~~~&~~u~~e~~~~~ Since the majority of non-pyramidal neurons are considered to represent inhibitory, GABAergic neurons, the demo~stmtion of bicueulline-sensitive

203

lPSPs suggests i~bibition of GABAergic neurons by GABAergic terminals. The existence of such a circuitry was investigated in both the hilar region and lhe stratum la~unosum-moie~~lar~ by GAD~mmun~yto~hem~stry~ In all ~p~~arnpal layers numerous cells were irnrn~o~in~d. In the hifns we noted a preponderance in the subgranular zone of the suprapyramidal blade of the fascia dentata. Three GAD-immunoreactive neurons are shown (Fig. 9a) in this characteristic position just below the granule cell

Bit

Fig. 7. Synaptic activation of a II&W~~n-pymrn~d~i neuron (A) and a granule ceI1(B) from perforrmt path in a transverse slice. The bottom trace of (I) is the response of the non-pyramidaf neuron to perforant Path stimulation which at the resting membrane potential (- 70 mV) seems to consist of an EPSP. ~epolar~~~~on by 15 mV of the ceil (top trace of 1) by the injection of constant direct current revealed that a hy~rp~la~z~n~ IPSP preceded the EPSP. In (2) both traces are superimposed. The point of divergence of the two traces after the stimulus artifact denotes the onset of the IPSP. In (3) and (4) the synaptic response is elicited during the injection of a long (170 ms) current pulse of +0.2nA (end of pulse not shown) which by itself triggers initially an action putential (4) taken after the slice had been bathed in bicucufline (25 PM) for 15 min. Note that the initial hyperpolarizatian fallowing the stimulus artifact has disappeared. (5) The response to the same stimulus as in (I) and (2) aRer the slice had been bathed in bicucuiline for 45 min. The response is now strongly excitatory. jB) Response of a granufe cell (resting membrane potential -70 mV) to perforanr path st~mu~at~o~ in central saline during h~~~l~r~za~~on by 1OmV (upper trace) and during depolarization by 1SmV (lower trace). 30th responses are superimposed as in (A2) to allow direct comparison. The EPSP is curtailed by an IPSP which becomes evident during depolarization. However, the point of divergence of the two traces is on the rising phase of the EPSP. Voltage calibratian is the same for ail traces of (A), time calibration in (2) applies also to (1), and the one in (4) to (3H5).

U.

204

MISGELD and

layer. In this particular zone, hilar non-pyramidal neurons were recorded and stained (Figs 3b,c and 8). Immunoreactive axon terminals form pericellular baskets around non-immunorea~tive cell bodies of granule cells. We also observed immunoreactive puncta surrounding the GAD-positive perikarya (arrowheads, Fig. 9a). Several immunoreactive neurons in the deep hifar region and in stratum radiatum and stratum lacunosum-mol~ulare of CA3 were processed for electron microscopy. In Fig. 9b, a GAD-immunoreactive terminal is seen which forms a symmetric synaptic contact on the cell body of a horizontal GAD-immunoreactive neuron located in the hilar region underneath the granule cell layer. Very similar observations were made in GADpositive neurons in stratum iacunosum-moleculare. We conclude that GABAergic neurons in both regions are innervated by GABAergic terminals.

DISCUSSION

We consider a main result of our study is that, in the hilar region subjacent to the granule cell layer, non-pyrami~i neurons have characteristics which they share with presumably inhibitory neurons in other hippocampal regions. Further, our study led US to conclude that GABAergic dis- inhibition, i.e. GABAergic inhibition of non-pyr~idal, pres~ably inhibitory GABAergic cells, plays a major role in information processing in the hippocampus because IPSPs could be elicited in all non-pyramidal neurons studied and because the IPSPs were bicucullinesensitive. Supportive evidence was provided by immunocytochemistry which showed GAD-immunoreactive terminals establishing symmetric synaptic contacts with GAD-positive non-pyramidal neurons in the hilar region and in stratum lacunosummoleculare. This may not be confined to these areas since GAD-positive synapses on GAD-positive cells have also been observed in the CAI region of the guinea-pig ~p~ampus.16 In previous studies9,22,37on non-pyramidal neurons in the hippocampus, it has been emphasized that these neurons share common electrophysiological chara~te~stics. This finding is somewhat su~rising in light of the fact that, in Golgi material, such neurons exhibit an enormous diversity. Yet when examined under the electron microscope, many non-pyramidal neurons share some common characteristics. Their perikarya are rather large, containing dense accumulations of endoplasmic reticulum. They exhibit varicose dendrites, which are densely covered with synaptic boutons mainly forming as~met~c synaptic contacts.33 We also observed some electrophysiological characteristics which hilar nonpyramidal cells have in common with non-pyramidal neurons in the hippocampus proper,” but not with pyramidal neurons and granule ceils. These concern their action potential waveform, their current-voltage

M.

FROTSCHER

relation and their ability to generate bursts m the presence of bicuculline. Of course, the argument on the diversity of nonpyramidal neurons is especially applicable to the hilar region. AmaraP has differentiated no less than 21 types of hilar neurons on the basis of Golgi impregnation. On the other hand, at least 60% of the neurons in the hilus were found to be GABAergic? Hence, a greater uniformity appears to exist with respect to the transmitter contained in these neurons than could be anticipated from Golgi material. Viewing the hilus as a whole, Liibbers ef al.” observed that a preponderance of GAD-immunoreactive cells was found in the region subjacent to the suprapyrdmidai blade of the granule cell layer (polymorphic Lone) which corresponds to the area we obtained our recordings from. It is reasonable, therefore, to assume that many, if not all neurons recorded and stained in the present experiments were GABAergic. In particular, all neurons stained in the hilus were located paraflel to the gran~ar layer. Thus, they closely resembled the horizontal type of GABAergic neuron3’.r8 In fact, in our immunocytochemical study cells of this type were also found to be GADimmunoreac~ve. One may further argue that the neurons in the hilar region are not the typical “interneurons” of the hippocampal formation. It is well-known that they provide the commissural innervation of the contralateral fascia dentata in addition to ipsilaterai associational fibers.*‘~24*47 However, there is also increasing evidence that many non-pyramidal neurons in stratum radiatum and stratum oriens of the hippocampus proper, commonIy regarded as “interneurons”, are projecting cells.2~14~‘7 GABAergic inhibition of GABAergic neurons will give rise to disinhibition of pyramidal and granule ceils. A particular function of disinhibition was suggested by our observation on hilar non-pyramidal neurons which inhibit granule cells, as is indicated by immunocytochemical findingsz3.” Perforant path activation induced first inhibition and then excitation in hilar non-pyramidal neurons (Fig. 7), the initial inhibition suppressing early excitation. This pattern of synaptic activation from perforant path is entirely different to the EPSP/IPSP sequences elicited in granule cells from the same stimulation site. Thus, the hilar neurons were inhibited at a time when granule cells were excited. This disinhibition may provide a gate for the excitation of granule cells via the perforant path, until the activation of non-pyramidal neurons through recurrent mossy fiber collaterals terminates this disinhibition.

thank Dr P. Stanton for reading the English manuscript. The authors also wish to thank Dr W. H. OerteI for providing the GAD antiserum and B. Weber and E. Thiefen for excellent technical assistance. This study was supported by the Deutsche Forschungsgemeinschaft (Fr 620/2-l, SFB 45 and SFB 220). Acknowlecigemenfs-We

Non-pyramidal

neurons

in the hippocampus

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